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Almost 50% of all cases of infertility may be associated with a male factor. Although a semen analysis has classically been used as the gold standard for determining a man’s fertility, this test may not detect abnormalities at the molecular level that may contribute to the 25% of cases that remain unexplained.
There is now growing evidence to support a link between oxidative stress and male infertility1,2. Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radicals (OH-) and superoxide anions (O2-) are generated by human sperm as part of their normal metabolism3- 5. At low levels, ROS enhance sperm capacitation and hyperactivation, as well as promoting the acrosome reaction and binding to the oocyte zona pellucida. ROS are maintained at low levels by effective anti-oxidant pathways, both within the sperm cytoplasm and more significantly in the seminal plasma, where high levels of ROS scavengers are found. However, if the production of ROS overwhelms the capacity of these anti-oxidant pathways to maintain appropriate low levels, then oxidative stress occurs, leading to pathological effects1-9. ROS initiates peroxidation of membrane lipids, proteins and DNA, which leads to the formation of potentially genotoxic and mutagenic adducts, damaging membrane function, ion gradients and receptor-mediated signal transduction as well as interfering with DNA methylation. This affects the fertilisation process1,10-12 as well as causing DNA fragmentation and gene mutations1,6,13,14. Manifestations of oxidative stress include semen parameter impairment, particularly a reduction in motility and vitality1,10. While cytoplasmic droplets retained on immature sperm are a source of excess ROS production, leukocytes in semen are up to a thousand fold more effective at generating ROS2,7,15. A list of the major exogenous sources of ROS are given in the table below.
Several studies show that there is a significant increase in ROS levels and a reduced antioxidant capacity in infertile men compared with fertile controls, irrespective of semen parameters2,4. Indeed, high levels of ROS have been observed in 30 – 80% of infertile men. High ROS levels impair semen parameters and fertilisation1,11,12, adversely affect blastocyst development and negatively affect pregnancy rates after IVF16. Furthermore, elevated seminal ROS levels are correlated with an increased time to natural conception2. One study showed an association between oxidative stress levels and recurrent miscarriage17.
Approximately 45% of male fertility patients are diagnosed with varicocoele, cryptorchidism, testicular torsion or endocrine imbalance. All of these conditions are associated with oxidative stress and impaired blood flow which in turn can increase germ cell apoptosis resulting in oligozoospermia18,19.
Large numbers of leukocytes are found in semen as a result of genitourinary tract infection or MAGI20,21. Sperm are vulnerable to ROS attack in the testicular environment as they are not in contact with the protective antioxidants of the seminal plasma and are particularly susceptible during epididymal transit – especially if there is inflammation resulting from infection.
The Reactive Oxygen Species (ROS) test is a relatively simple chemiluminescence test requiring a fresh semen sample produced by masturbation after 2 – 3 days sexual abstinence. Measurement of ROS is performed in whole semen within 15 - 30 minutes of ejaculation. ROS are measured indirectly using a probe such as luminol, which is oxidised in the presence of ROS, resulting in chemiluminescence. The luminescence generated by this reaction is measured using a luminometer. The result can be reported within 24 hours. The test can be requested alone or in conjunction with a semen analysis or any other test for male reproductive health. The test has been fully validated and is CE marked22,23.
Identification of infertile patients who demonstrate oxidative stress in their semen may assist in the management of male infertility. Increased levels of free radicals may be reduced with a change in lifestyle and a diet rich in anti-oxidants, designed to protect against oxidative stress13,14,24,25. Randomised placebo controlled studies have shown that oral anti-oxidant treatment can decrease seminal ROS levels and sperm DNA damage, and improve pregnancy rates14,25. Varicocoele is a major cause of male infertility. Current evidence shows that clinical varicocoele repair not only significantly improves semen parameters and reduces oxidative stress and DNA damage26,27, but also increases pregnancy rates, both naturally28 and with assisted conception29,30. Thus information about oxidative stress levels in men with varicocoele may aid urologists in the decision to perform varicocoele repair. Treatment of infections would also be expected to reduce ROS levels21. A large randomised study compared men with Chlamydia or Ureaplasma infection with and without antibiotics for 3 months21. Those treated showed a significant fall in ROS levels, improved sperm motility and a significant increase in pregnancy rates. Initiatives to reduce the levels of ROS can be assessed by undertaking a second test three months after the first. If levels remain high, a sperm DNA fragmentation test may be considered.
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2. Tremellen, K (2008) Oxidative stress and male infertility: a clinical perspective. Hum. Reprod. Update 14, 243–258.
3. Du Plessis SS, Agarwal A, Halabi J and Tvrda E (2015) Contemporary evidence on the physiological role of reactive oxygen species in human sperm function. J Assist Reprod Genet 32:509–520.
4. Aitken RJ (1995) Free radicals, lipid peroxidation and sperm function. Reprod fertile Dev 7(4):659-68.
5. Aitken RJ, Gibb Z, Baker MA, Drevet J, Gharagozloo P (2016) Causes and consequences of oxidative stress in spermatozoa. Reprod Fertil Dev 28(1-2):1-10.
6. Menezo YJR, Silvestris E, Dale B, Elder K (2016) Oxidative stress and alterations in DNA methylation: two sides of the same coin in reproduction RBMOnline 33: 668–683.
7. Henkel RR (2011) Leukocytes and oxidative stress: dilemma for sperm function and male fertility. Asian J Androl 13: 43–52.
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9. Gosalvez J, Tvrda E, Agarwal A. (2017) Free radical and superoxide reactivity detection in semen quality assessment: past, present, and future. J Assist Reprod Genet. 2017 Mar 25. doi: 10.1007/s10815-017-0912-8. [Epub ahead of print]
10. Morielli T, O'Flaherty C (2015) Oxidative stress impairs function and increases redox protein modifications in human spermatozoa. Reproduction 149(1):113-23.
11. Ghaleno LR, Valojerdi MR, Hassani F, Chehrazi M, Janzamin E (2014) High level of intracellular sperm oxidative stress negatively influences embryo pronuclear formation after intracytoplasmic sperm injection treatment. Andrologia 46:1118-27.
12. Chen SJ, Allam JP, Duan YG, Haidl G. (2013) Influence of reactive oxygen species on human sperm functions and fertilizing capacity including therapeutical approaches. Arch Gynecol Obstet 288:191-9.
13. Wright C, Milne S, Leeson H (2014) Sperm DNA damage caused by oxidative stress: modifiable clinical, lifestyle and nutritional factors in male infertility. Reprod Biomed Online 28: 684-703.
14. Gharagozloo P and Aitken RJ (2011) The role of sperm oxidative stress in male infertility and the significance of oral antioxidant therapy. Hum Reprod. 2011 Jul;26(7):1628-40. Review.
15. Mupfiga C, Fisher D, Kruger T, Henkel R (2013) The relationship between seminal leukocytes, oxidative status in the ejaculate, and apoptotic markers in human spermatozoa. Syst Biol Reprod Med 59: 304-11.
16. Zorn B, Vidmar G and Meden-Vrtovec H (2003) Seminal reactive oxygen species as predictors of fertilization, embryo quality and pregnancy rates after conventional in vitro fertilization and intracytoplasmic sperm injection. Int. J Androl. 26 (5): 279-85.
17. Imam SN, Shamsi MV, Kumar K, Deka D and Dada R (2011) Idiopathic recurrent pregnancy loss: role of paternal factors; a pilot study J Reprod Infertil 12(4): 267 – 276.
18. Turner TT and Lysiak JJ (2008) Oxidative stress: a common factor in testicular dysfunction. J Androl. 2008 Sep-Oct;29(5):488-98.
19. Hamada A, Esteves SC, Agarwal A (2013) Insight into oxidative stress in varococele-associated male infertility: part 2. Nat Rev Urol 10 :26-37.
20. Potts Jm, Pasqualotto FF (2003) Seminal oxidative stress in patients with chronic prostatitis. Andrologia 35(5):304-8.
21. Vicari E (2000) Effectiveness and limits of antimicrobial treatment on seminal leukocyte concentration and related reactive oxygen species production in patients with male accessory gland infection. Hum Reprod. 15(12): 2536-44.
22. Vessey W, Perez-Miranda A, Macfarquhar R, Agarwal A, Homa S. (2014) Reactive oxygen species (ROS) in human semen: validation and qualification of a chemiluminescence assay. Fertil Steril. 102:1576-1583.
23. Homa ST, Vessey W, Perez-Miranda A, Riyait T, Agarwal A (2015) Reactive oxygen species (ROS) in human semen: determination of a reference range. J Assist Reprod Genet 32(5):757-64.
24. Greco E, Romano S, Iacobelli M, Ferrero S, Baroni E, Minasi MG, Ubaldi F, Rienzi L, Tesarik J (2005) ICSI in cases of sperm DNA damage: beneficial effect of oral antioxidant treatment. 20(9):2590-4.
25. Showell MG, Mackenzie-Proctor R, Brown J, Yazdani A, Stankiewicz MT and Hart RJ (2014) Antioxidants for male subfertility. Cochrane Database Syst Rev Dec 15;(12):CD007411.
26. Dada R, Shamsi MB, Venkatesh S, Gupta NP and Kumar R (2010) Attenuation of oxidative stress & DNA damage in varicocelectomy: implications in infertility management. Indian J Med Res 132: 728 – 730.
27. Ni K, Steger K, Yang H, Wang H, Hu K, Zhang T, Chen B (2016) A comprehensive investigation of sperm DNA damage and oxidative stress injury in infertile patients with subclinical, normozoospermic, and astheno/oligozoospermic clinical varicocoele. Andrology 4(5):816-24.
28. Tiseo BC, Esteves SC, Cocuzza MS (2016) Summary evidence on the effects of varicocele treatment to improve natural fertility in subfertile men. Asian J Androl. 18(2):239-45.
29. Esteves SC, Roque M, Agarwal A (2016) Outcome of assisted reproductive technology in men with treated and untreated varicocele: systematic review and meta-analysis. Asian J Androl. 18(2):254-8.
30. Kirby EW, Wiener LE, Rajanahally S, Crowell K, Coward RM (2016) Undergoing varicocele repair before assisted reproduction improves pregnancy rate and live birth rate in azoospermic and oligospermic men with a varicocele: a systematic review and meta-analysis. Fertil Steril 106(6):1338-1343.